In this study, three epigallocatechin gallate glycosides were synthesized by the acceptor reaction of a glucansucrase produced by Leuconostoc mesenteroides B-1299CB with epigallocatechin gallate (EGCG) and sucrose. Each of these glycosides was then purified, and the structures were assigned as follows: epigallocatechin gallate 7-O-alpha-D-glucopyranoside (EGCG-G1); epigallocatechin gallate 4'-O-alpha-D-glucopyranoside (EGCG-G1'); and epigallocatechin gallate 7,4'-O-alpha-D-glucopyranoside (EGCG-G2). One of these compounds (EGCG-G1) was a novel compound. The EGCG glycosides exhibited similar or slower antioxidant effects, depending on their structures (EGCG > or = EGCG-G1 > EGCG-G1' > EGCG-G2), and also manifested a higher degree of browning resistance than was previously noted in EGCG. Also, EGCG-G1, EGCG-G1', and EGCG-G2 were 49, 55, and 114 times as water soluble, respectively, as EGCG.

Alkyl glucosides were synthesized by the reaction of Leuconostoc mesenteroides dextransucrase with sucrose and various alcohols. Alkyl alpha-D-glucosides were obtained with a yield of 30% (mol/mol) with primary alcohols, but secondary alcohols or tertiary alcohols gave yields below 5%. The optimal yield was 50% using 1-butyl alpha-D-glucoside with 0.9 M 1-butanol. The acceptor products of methanol or ethanol were confirmed as methyl alpha-D-glucopyranoside and ethyl alpha-D: -glucopyranoside via MALDI-TOF MS and NMR analysis. Thus, methyl or ethyl alpha-D-glucoside constituted half the emulsification activities of Triton X-100 as commercially available surfactants.

Recombinant heterologous proteins can be produced as insoluble aggregates partially or perfectly inactive in Escherichia coli. One of the strateges to improve the solubility of recombinant proteins is fusion with a partner that is excellent in producing soluble fusion proteins. To improve the production of soluble β-galactosidase, the gene of Thermus thermophilus KNOUC112 β-galactosidase (KNOUC112 β-gal) was fused with thioredoxin gene, and optimization of its expression in E. coli TOP10 was performed. KNOUC112 β-gal in pET-5b was isolated out, fused with thioredoxin gene in pThioHis C, and transformed to E. coli TOP10. The β-galactosidase fused with thioredoxin was produced in E. coli TOP10 as dimer and trimer. The productivity of fusion βgalactosidase expressed via pThioHis C at 37°C was about 5 times higher than that of unfused β-galactosidase expressed via pET-5b at 37°C. Inclusion body of β-galactosidase was formed highly, regardless of the induction by IPTG when KNOUC112 β-gal was expressed via pET-5b at 37°C. Fusion β-galactosidase expressed at 37°C via pThioHis C without the induction by IPTG was soluble, but the induction by IPTG promoted the formation of inclusion body. Lowering the incubation temperature for the expression of fusion gene under 25°C prevented the formation of inclusion body, optimally at 25°C. 0.07 mM of IPTG was sufficient for the soluble expression of fusion gene at 25°C. The soluble production of Thermus thermophilus KNOUC112 β-galactosidase could be increased about 10 times by fusion with thioredoxin, and optimization of incubation temperature and IPTG concentration for induction.

A gene (lsd1) encoding dextranase from Lipomyces starkeyi KSM22 has been previously cloned, sequenced, and expressed in Saccharomyces cerevisiae. The gene consisting of 1,824 base pairs and encoding a protein of 608 amino acids was then cloned into and secretively expressed in Pichia pastoris under the control of the AOX1 promoter. The dextranase productivity of the P. pastoris transformant (pPIC9K-LSD1, 134,000 U/l) was approximately 4.2-fold higher than that of the S. cerevisiae transformant (pYLSD1, 32,000 U/l) cultured in an 8-l fermentor. Over 0.63 g/l of active dextranase was secreted into the medium after methanol induction. The dextranase of the P. pastoris transformant, as analyzed by SDS-PAGE and Western blotting, showed only one homogeneous band. This dextranase of the P. pastoris transformant showed a broad band near 73 kDa. Rabbit monoclonal antibodies against a synthetic LSD1 peptide mix also recognized approximately 73 kDa.